title: Redesigning the active-site of an acyl-CoA dehydrogenase: new evidence supporting a one-base mechanism.
abstract: The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homology and a common catalytic mechanism which involves abstraction of an alpha-proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E2 site), but is in a different location in two other family members (the E1 site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E2 to the E1 site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme (K(m) = 0.6 and 2.0 microM, and Vmax = 2.8 and 0.3 s-1 for native and mutant enzyme respectively). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis--abstraction of the alpha-HR from the substrate followed by transfer of the beta-HR to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E1 and E2 sites are topologically similar and catalytically competent. However, analysis of the 1H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform gamma-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the alpha-HR but is far removed from the gamma-Hs. These findings provide further support for a one base mechanism of alpha- and gamma-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
authors.forename: S
authors.initials: S
authors.lastname: Dakoji
authors.forename: I
authors.initials: I
authors.lastname: Shin
authors.forename: K P
authors.initials: KP
authors.lastname: Battaile
authors.forename: J
authors.initials: J
authors.lastname: Vockley
authors.forename: H W
authors.initials: HW
authors.lastname: Liu
mesh_terms: D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D051381:Rats; D016415:Sequence Alignment; D013237:Stereoisomerism
chemical_list: D044944:Acyl-CoA Dehydrogenases